p53 is the most commonly mutated gene in human cancers. Recently, two related genes, p73 and p63, have been described which are capable of transactivation and cell-death inducing activities typically associated with the p53 tumor suppressor. Nevertheless, the physiological functions of either gene remain largely unknown. This is a proposal to uncover the function of the p63 gene, and its potential role in processes of cell growth regulation, mammalian development, and tumorigenesis. Importantly, p63 is highly expressed in the basal, or proliferative, cell populations of a wide range of epithelial tissues that are frequent sites of carcinoma in humans. These include skin, cervical and urogenital, epithelia, as well as the prostate and breast. As one means of probing p63 function, we have analyzed the effects of targeted disruptions of this gene in mice. These mice show profound defects in all squamous epithelial, as well as dramatic limb and craniofacial abnormalities. The analysis of p63 is complicated by the existence of multiple isotypes that exhibit strikingly different properties. One class of p63 proteins show functional similarities to p53, while the other appears to have opposite, even dominant-negative activities. An important goal in this proposal is to distinguish these p63 isotypes and determine their physiological significance using murine models. A second focus is to understand the mechanistic basis of p64's essential role, evident from our p63-deficient mice, in regulating the proliferative capacity and differentiation of epithelial cells. Thirdly, as p53 function is tightly controlled by its protein stability and post-translational mechanisms, we will determine if similar mechanisms regulate p64. Finally, we will use differential expression assays to identify target genes of transactivating and suppressing p63 isotypes. We anticipate that the proposed studies will yield new insights toward the function of p63 in regulating epithelial cell proliferation and maturation, and its role in development and tumorigenesis.